Comprehensive Cross-Linking Mass Spectrometry Reveals Parallel Orientation and Flexible Conformations of Plant HOP2–MND1

The HOP2–MND1 heterodimer is essential for meiotic homologous recombination in plants and other eukaryotes and promotes the repair of DNA double-strand breaks. We investigated the conformational flexibility of HOP2–MND1, important for understanding the mechanistic details of the heterodimer, with chemical cross-linking in combination with mass spectrometry (XL–MS). The final XL–MS workflow encompassed the use of complementary cross-linkers, quenching, digestion, size exclusion enrichment, and HCD-based LC–MS/MS detection prior to data evaluation. We applied two different homobifunctional amine-reactive cross-linkers (DSS and BS²G) and one zero-length heterobifunctional cross-linker (EDC). Cross-linked peptides of four biological replicates were analyzed prior to 3D structure prediction by protein threading and protein–protein docking for cross-link-guided molecular modeling. Miniaturization of the size-exclusion enrichment step reduced the required starting material, led to a high amount of cross-linked peptides, and allowed the analysis of replicates. The major interaction site of HOP2–MND1 was identified in the central coiled-coil domains, and an open colinear parallel arrangement of HOP2 and MND1 within the complex was predicted. Moreover, flexibility of the C-terminal capping helices of both complex partners was observed, suggesting the coexistence of a closed complex conformation in solution